Often in the analysis of aqueous fluids for chemical or biological substances (herein called analytes), the results of the analysis can be adversely affected by interfering materials. These materials either interfere with the reactivity of the analyte or act so similarly to it that the properties of analyte and interferent cannot be distinguished. For example, in the determination of certain isoenzymes, it is often necessary to minimize or eliminate the effect of the isoenzymes which are not of interest.
Creatine kinase (abbreviated herein to CK, but also known as creatine phosphokinase, CPK, or ATP: creatine phosphotransferase E.C.2.7.3.2.) occurs in human body fluids and tissue in the form of three isoenzymes: CK-MM, for example in muscles, CK-BB, for example in the brain, and CK-MB, for example in the myocardium. The CK activity occurring in healthy human blood serum is normally due to the CK-MM isoenzyme because CK-BB does not generally pass into the blood stream. In a healthy individual, CK-MB is generally restricted to certain organs, for example the myocardium. However, when the myocardium is damaged, as in the case of a cardiac infarction, CK-MB is released into the blood stream and can be detected therein.
A potential difficulty encountered in methods for determining CK-MB in biological fluids is interference from the other two isoenzymes. For practical purposes, the amount of CK-BB in the fluid is considered negligible in most determinations. In methods for determining CK-MB, it is known to precipitate or inhibit the M subunit with specific antibodies to eliminate the interference of CK-MM on the assay and then to measure the remaining isoenzyme CK-MB.
A relatively recent contribution to clinical chemistry was the development of dry multilayer analytical elements useful for the assay of liquids. Such elements are described, for example, in U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al). These elements generally have an outer spreading layer which is known as a porous "blush" polymer layer (see Col. 7, lines 44-64) composed of a polymeric binder and a particulate material which increases layer porosity.
A number of analytical elements having such spreading layers have been designed for various assays and used commercially, including an element useful for the determination of total CK. In the preparation of such elements, it has been standard practice to incorporate some reagents into the spreading layer by means of a wash coat. In other words, the spreading layer is coated and dried, and an aqueous suspension of the reagent(s) is applied to it and allowed to soak into the spreading layer. The aqueous medium carries the reagent(s) throughout the spreading layer.
It has been attempted to incorporate antibodies for CK-MM into a spreading layer in this manner (i.e. as a wash coat). However, it has been observed that the resulting element has reduced stability in high humidity environments, i.e. at least about 50% relative humidity at 25.degree. C. Moreover, manufacturing processes used in making analytical elements are necessarily carried out in this type of environment. The reduced stability under such conditions is a serious problem. It limits the flexibility in handling the elements by both the manufacturer and the user. The manufacturer must try to limit the amount of time the element is subject to high humidity. Further, if a user accidentally leaves the element out of the freezer compartment it is normally kept in prior to use, the element is likely to give erroneous results in the assay.
It would be desirable to improve the stability, and hence the room temperature keeping properties, of analytical elements containing active antisera which have blush polymer spreading layers like those described in the Przybylowicz et al reference noted above.